7,8-Dihydroxy Flavone Induces Apoptosis via Upregulation of Caspase-3 in Human Hepatocarcinoma Cell

Objective: 7,8-Dihydroxyflavone, a tyrosine kinase receptor agonist, is a flavonoid that has recently gained the attention of researchers due to its anticancer properties. Nevertheless, molecular pathways of 7,8-dihydroxyflavone for hepatocarcinoma are uncertain. Our aim was to identify the impact of 7,8-dihydroxyflavone on human hepatocarcinoma. Material and Methods: Human hepatocarcinoma cell line-7 cells were used as human hepatocarcinoma cells, and 7,8-dihydroxyflavone was applied to the cells at various doses. The cytotoxic and apoptotic effects of 7,8-dihydroxyflavone were determined with Alamar Blue and flow cytometry. The properties of 7,8-dihydroxyflavone on the mRNA expressions related with Bcl-2, Bax, cleaved-caspase-3 genes, and protein expressions were determined via quantitative real-time polymerase chain reaction and western blot analysis, respectively. Results: 7,8-Dihydroxyflavone-enhanced cell death in human hepatocarcinoma cell line-7 via the overexpression of cleaved-caspase-3 (P = .003) and decreased Bcl-2 (P = .038) protein levels. Furthermore, cleaved-caspase-3 mRNA overexpression (P = .001) markedly led to 7,8-dihydroxyflavone-induced apoptosis. Conclusion: 7,8-Dihydroxyflavone could promote apoptotic cell death by modulating caspase pathways and suppressing antiapoptotic protein. These characteristics may mediate to clinical practice of 7,8-dihydroxyflavone for prevention and therapy of hepatocarcinoma.


Introduction
Hepatocellular carcinoma (HCC) is the most common diagnosis of liver cancer-related deaths.Surgery, chemo-or radiotherapy are treatment options according to the stage of the tumor.Besides that, the evolvement of promising treatment alternatives is needed for effective therapy.6][7] Flavonoids are sorted into different subtypes such as flavones, flavonols, flavanones, flavanonols, isoflavones, and flavanols due to the scavenging capacities of free radicals. 36][7][8] The effects are primarily focused on cancers, 3,4,9 Alzheimer' s disease (AD), 10,11 atherosclerosis, 12,13 and biochemical pathways. 14,15These compounds could affect many biological functions by scavenging free radicals, stimulating apoptosis and signal transduction pathway or inhibiting proliferation in human cancer cells. 6,1517]18 Also, it has neurotrophic effects.Besides that, 7,8-DHF presents antioxidant capacity by inhibiting glutamate-stimulated, 6-hyd roxydopami ne-st imula ted dopaminergic neurotoxicity, and oxidative stress-stimulated genotoxicity. 197,8-Dihydroxyflavone has protective effects by downregulation of nuclear factor erythroid 2-related factor 2 and Heme oxygenase 1 in the liver. 178-Dihydroxyflavone was previously associated with Alzheimer's and Parkinson's diseases. 10,11A structure-function connection study demonstrated that the catechol part in 7,8-DHF is fragile for agonistic effect; nevertheless, the catechol part is commonly metabolized by methylation, glucuronidation, sulfation in the liver, and this leads to limited oral bioavailability. 16,17e inhibitory properties of 7,8-DHF in various cancer cell lines have been demonstrated. 15,20ark et al 15

Cell Cultures
The human hepatocarcinoma cancer cell lines, human hepatocarcinoma cell line-7 (HUH-7), were kindly provided by Department of Biological Sciences, Middle East Technical University.Ethics committee approval is no longer needed for cell culture studies.Cells were grown in Dulbecco' s modified Eagle' s medium (DMEM; Biological Industries, Cromwell, Conn, USA) containing 10% fetal bovine serum (FBS; Biological Industries, Cromwell, Conn, USA), 1% penic illin -stre ptomy cin (Biological Industries, Cromwell, Conn, USA), and 2 mM l-glutamine (Biological Industries, Cromwell, Conn, USA) in a 37 o C, 5% CO 2 incubator.Approximately 85% of the passaged cells in the experiment were removed via trypsin.

Cell Viability Assays
Human hepatocarcinoma cell line-7 cells were cultured at 1 × 10 4 cells/mL into plates and incubated for a day.The cells were exposed to several dosages of 7,8-DHF, ranging from 1-300 μM and incubated at 37°C for 48 hours to find out the IC 50 (Inhibitory Concentration) value.Cell viability was determined from the absorbances at 520 and 620 nm (Multiskan Go; Thermo Scientific Co., Waltham, Mass, USA) by AlamarBlue assay 21

Quantitative Real-Time Polymerase Chain Reaction Analyses
RNA was isolated by the Trizol method. 25uman hepatocarcinoma cell line-7 cells were transfected with a concentration equivalent to IC 50 to 7,8-DHF for 48 hours.The concentration and purity of the RNA were specified by evaluation of the absorbance at 260 and 280 nm via NanoDrope 1000 Spectrophotometer.cDNA (iScript cDNA Synthesis Kit; Bio-Rad) from the RNA population was synthesized.cDNA analysis was performed by reverse transcribing 1 µg of total RNA into cDNA.Bax, Bcl-2, cleaved-caspase-3, and GAPDH levels were analyzed via quantitative real-time polymerase

Main Points
• No studies have been reported about the molecular pathways responsible for the impacts of 7,8-dihydroxyflavone (7,8-DHF) on hepatocellular carcinoma.
• The cytotoxic effects of 7,8-DHF on human hepatocarcinoma cell line-7 (HUH-7) cells were determined with Alamar Blue.The effects of 7,8-DHF on the mRNA and protein expressions were analyzed by quantitative real-time polymerase chain reaction and western blot, respectively.
• Cleaved-caspase-3 expression enhances tumor formation and is a critical step in the continuation and evolvement of cancer.7,8-Dihydroxyflavone and associated molecules may have significant molecules for cancer therapy.chain reaction (qRT-PCR).The target gene expressions (2 −∆∆Ct ) were normalized to the endogenous GAPDH gene expression. 26

Statistical Analysis
The The cell viability in HUH-7 was performed after 7,8-DHF therapy (Figure 1B).Besides, the growth of HUH-7 cells was specified.Treatment day was admitted as day 1. 7,8-Dihydroxyflavone considerably demonstrated its effect on cell growth on day 2. Results revealed that 7,8-DHF exhibited high cytotoxicity with the IC 50 value 177.6 μM at the 48th hour in HUH-7 (Figure 1D).In the next steps of the study, the cells were treated with the IC 50 dose of 7.8 DHF.

Discussion
The major molecular construction of a flavonoid includes 2 aromatic rings (A and B) attached by pyran-4-one ring C and roughly 4000 flavonoids found on earth. 3,6The position of the double bond in the C ring is proposed to be significant determinants for flavonoids' physiological activities.Otherwise, hydroxyl groups at C ring and the count of hydroxyl groups at B ring play a crucial role in the activity of flavonoids.It was suggested that the multifunctional impressions of flavonoids were due to structural and functional groups. 3,68-Dihydroxyflavone is a potent TrkB agonist in several disorders. 27,287,8-Dihydroxyflavone was notified to exhibit significant neurotrophic and antioxidant capacity. 6,28-307,8-Dihydroxyflavone was previously defined as a possible anticancer agent. 19Besides, the exact linkage of the anticarcinogen properties of 7,8-DHF is uncertain.Small numbers of flavonoids were presented to inhibit cell proliferation by triggering apoptosis in hepatocarcinoma cell lines. 3,4,6,9,17Despite the prohibitory effect of 7,8-DHF in certain kinds of carcinoma, 19,20,31 no data were reported about hepatocarcinoma yet.According to the results of our study, 7,8-DHF seems to inhibit   34 Cleaved-caspase-3 is a protein that interacts with caspase-8 and caspase-9 and plays a role in apoptosis.In the intrinsic pathway, cytochrome c released from mitochondria works with caspase-9, apoptosis-activating factor 1 (Apaf-1) to produce procleaved-caspase-3 with the availability ATP (Adenosine triphosphate). 35is study' s findings remarked that 7,8-DHF application led to the occurrence of apoptosis.
Apoptosis was associated with increased levels of cleaved-caspase-3, overexpression of Bax, and decreased Bcl-2, which play roles in the initiation and regulation of the apoptosis process. 31hese results suggest that 7,8-DHF-induced apoptosis via caspase-dependent mechanism by disrupting the cell death on HUH-7 cells compared to controls.
Limitations, drawbacks, or shortcomings: In this study, cytotoxicity and cell death analyses were performed with adequate and reliable methods.To understand the metabolic pathways of cell death, apoptotic and antiapoptotic proteins, experiments were performed with protein and mRNA steps.Notwithstanding, further protein analyses could be performed such as p53, caspase 8-9, Apaf-1, and additional cell cycle analyses could be performed to confirm the results.
In conclusion, a large number of studies analyzing novel anticancer agents and biomarker compounds that are considered useful for cancer treatment should be concentrated particularly on cellular mechanisms such as apoptosis, expression of protein and mRNA.The cleaved-caspase-3 expression enhances tumor formation and is a critical step in the continuation and evolvement of cancer.According to these results, 7,8-DHF inhibits the proliferation of HUH-7 cells by affecting programmed cell death.These new phenomena were not already defined for 7,8-DHF, and given that 7,8-DHF and associated molecules may have significant roles as agents for cancer therapy.To the best of our knowledge, we have described the molecular mechanisms of the effect of 7,8-DHF for the first time in HUH-7 cells.7,8-Dihydroxyflavone might be a gradually impressive suppressant or remedial substance in tumor genesis.
concluded that 7,8-DHF causes cell death of leukemia cells by blocking G1 phase in the cell cycle.Lee et al demonstrated that 7,8-DHF could reorganize Sp1 activity and trigger apoptosis by modulating the cell cycle and diminishing antiapoptotic protein levels.No researchers have reported the molecular pathways responsible for these impacts on HCC.Our aim was to examine the cytotoxic and apoptotic effects of 7,8-DHF including cell viability, apoptosis, and cell cycle-related protein and gene expressions on human hepatocarcinoma cells.

Figure 2 .
Figure 2. A-D.Human hepatocarcinoma cell line-7 (HUH-7) cells were treated with 177.6 μM 7,8-dihydroxyflavone (7,8-DHF) for 48 hours.(A) Effect of 7,8-DHF on apoptosis-induced cell death examined by flow cytometry following Annexin V-APC and 7-AAD staining.Western blotting was performed to analyze the Bax, Bcl-2, and caspase-3.GAPDH was used as an internal control and applied for normalization.(B) Representative immunoblot of HUH-7 cell protein in experimental control and 7,8-DHF-treated groups.(C) Analysis of band density of protein expression of the control and 7,8-DHFtreated groups.(D) Alterations in mRNA expressions were analyzed by using quantitative real-time polymerase chain reaction.Control and 7,8-DHF groups were presented with dark and light colors, respectively.Results are given as mean ± SD (statistically significant difference vs. control: * P < .05,** P < .01. Statistical Package for the Social Sciences (SPSS) 21.0 (IBM SPSS Corp.; Armonk, NY, USA)) was used for statistical analysis.The Student' s t-test and Mann-Whitney U test were performed for comparison.Values are expressed as mean ± SD.Three replicates were done.P-values less than .05were considered statistically significant.
Results7,8-Dihydroxyflavone Showed Cytotoxic Properties on Hepatocarcinoma Cells Alamar Blue was conducted to find out cytotoxic activities of 7,8-DHF on HUH-7.IC 50 was calculated by sigmoidal plot analysis using GraphPad.